1. Field of the Invention
This invention relates generally to angeogenesis and, more particularly, to anti-angeogenic polypeptides which are related to plasminogen and to methods of making and using such polypeptides.
2. Description of the Related Art
Angiogenesis, i.e. the formation of new blood vessels, involves a complex coordination of endothelial cell proliferation, migration, basement membrane degradation and neovessel organization (Ji et al., FASEB J. 12:1731-1738, 1998). During normal processes such as organogenesis in the embryo and wound healing in the adult, angiogenesis provides the necessary vascular support for the newly developing tissue. In pathologic conditions, however, the growth of new blood vessels can lead to advancement of disease processes including the development and progression of cancerous tumors, diabetic retinopathy, tissue and organ malformation, and cardiovascular disorders (Folkman, Nat. Med. 1:27-31, 1995). With respect to cancerous tumor, increasing evidence suggests that tumor growth and lethality are dependent upon angiogenesis and that angiogenesis inhibition suppresses tumor development (Folkman, Forum (Genova) 9:59-62, 1999; Folkman, Adv. Cancer. Res. 43:175-203, 1985).
A number of angiogenesis inhibitors, i.e. anti-angiogenic substances have been identified including angiostatin, thrombospondin, and glioma-derived angiogenesis inhibitory factor (Folkman, 1995 supra). Of these, angiostatin, a 38 kDa fragment of plasminogen, has been shown to have potent anti-angiogenic activity which inhibits tumor growth (O'Reilly et al., Nat. Med. 2:689-692, 1996; O'Reilly et al., Cell 79:315-328, 1994). This is in contrast to the full length plasminogen which has no anti-angiogenic activity (Id.).
Angiostatin contains the first four of plasminogen's five triple disulfide-linked loops known as kringle regions. The four kringle regions of angiostatin and the fifth kringle region found in plasminogen, but not angiostatin, have been characterized as to anti-angiogenic activity by studying the activities of various fragments of plasminogen. Kringle 5 fragments of plasminogen obtained by proteolysis of plasminogen and by recombinant techniques have been reported to exhibit potent endothelial-cell anti-proliferative activity (Cao et al., J. Biol. Chem. 272:22924-22928, 1997). Similarly, a recombinant kringle 1 fragment and a recombinant kringle 3 fragment have been shown to have potent anti-proliferative activity whereas a kringle 2 fragment shows much less inhibitory activity and a kringle 4 fragment exhibits markedly low inhibitory activity (Cao et al., J. Biol. Chem. 271:29461-29467, 1996; U.S. Pat. No. 6,024,688 to Folkman et al.). In contrast, a fragment containing kringles 2 and 3 showed only low activity, whereas a larger angiostatin fragment containing kringle regions 1-3 showed more potent anti-proliferative activity than angiostatin itself which contains kringle regions 1-5 (Id.).
Thus, it would appear that the anti-angiogenic activity of plasminogen fragments containing more than one kringle region cannot be accurately predicted based upon the activities of the individual kringle regions and, hence, the earlier studies provide no clear guidance as to whether additional fragments of plasminogen containing more than one kringle region will show anti-angiogenic activity. In addition, angiostatin itself was isolated under reducing conditions such that this substance is believe to a non-naturally occurring fragment as are the proteolytic and recombinant fragments reported in the earlier studies cited above. Such non-naturally occurring polypeptides could potentially elicit immunologic responses in a subject or other undesirable side effects which might be avoided with a naturally occurring fragment. Furthermore, the three dimensional structure of a naturally occurring fragment of plasminogen which functions as an endogenous angiogenic agent may not be accurately predicted from the three dimensional structure of the reported proteolytic or recombinant fragments in earlier reports. It would, thus, be desirable to identify and isolate new, naturally occurring polypeptide fragments of plasminogen which possess potent and efficacious anti-angiogenic activity and which have little or no potential for producing immunologic and other side effects.